Bacteria have a need to maintain their genomic integrity and defend against invading viruses and plasmids. Recently, genomic loci with clustered, regularly interspaced, short palindromic repeats (CRISPRs) were found in bacteria and were shown to mediate adaptive immunity to invading pathogens [1]: Bacteria can capture short nucleic acid sequences from invading pathogens and integrate them in the CRISPR loci. Small RNAs, produced by transcription of the CRISPR loci, can guide a set of bacterial endonucleases to cleave the genomes of invading pathogens.
The minimal requirements for one bacterial endonuclease, CAS9 from Streptococcus pyogenes, were characterized by purifying the enzyme and reconstituting the cleavage reaction in vitro [2]. Surprisingly, CAS9 itself is sufficient for endonuclease cleavage and no further polypeptides are required for the cleavage reaction. In addition, CAS9 requires two RNA cofactors: a constant tracrRNA and a crRNA bearing both constant and variable parts. Importantly, the variable part of the crRNA can be used to reprogram the cleavage specificity of CAS9, thereby enabling the targeting of CAS9 to genomic loci of interest. Cleavage specificity is limited by the protospacer adjacent motif (PAM) that is specific to CAS9 and lies adjacent to the cleavage site. In an attempt to simplify the system, crRNA and tracrRNA were fused to give rise to one chimeric RNA molecule referred to as the guide RNA.
Following this publication, several laboratories showed that this system can be used in cells from different species including humans [3,4], zebrafish [5], fruit flies [6] and yeast [7]. Once a certain locus is specified by the crRNA, CAS9 induces DNA double-strand breaks with remarkable efficiency at that particular locus and triggers cellular DNA damage repair mechanisms: In the presence of a homology template, homology-directed repair (HDR) allows the precise engineering of the locus of interest, enabling for instance the introduction of tags or point mutations. In the absence of a homology template, non-homologous end joining (NHEJ) is the predominant repair mechanism. As NHEJ is error-prone, it often creates small insertions or deletions. If the CAS9 cleavage site is located in an exon of a human gene, NHEJ often gives rise to frameshift mutations, thereby disrupting the gene of interest and generating a gene knockout.
The endonuclease CAS9 has two domains with endonuclease activity, a RuvCI domain and an HNH domain. Point mutations in either of the two domains generate a CAS9 nickase that cleaves only one of the two DNA strands, giving rise to nicked DNA [2]. This is of particular interest because nicked DNA is a suitable template for HDR, but not NHEJ [4]. So by using the CAS9 nickase, one can enhance HDR efficiency considerably. In addition, the introduction of two DNA nicks in close proximity can enhance cleavage specificity considerably [8].
Of note, CAS9 can not only be used to induce cleavage of a particular genomic locus, but it can be used as a universal targeting tool. For that purpose, catalytically inactive mutants of CAS9 turned out to be useful. For instance, fusion of inactive CAS9 to transcriptional activator domains enables the targeted activation of transcription [9]. Using this approach, a plethora of applications of CAS9 is conceivable in which CAS9 serves as targetable genome tether.
Finally, variants of CAS9 have been identified in several other bacterial species including Streptococcus thermophiles, Neisseria meningitis and Treponema denticola [10,11]. Importantly, all CAS9 variants described so far differ with regard to their PAM, thereby enlarging the repertoire of accessible cleavage sites and enabling the simultaneous targeting of several sites by orthogonal CAS9 proteins.
US 2010/0076057 A1 discloses the targeted DNA interference with crRNA and CRISPR-associated (cas) proteins, in particular for horizontal gene transfer based on the use of CRISPR sequences.
The RNA-directed DNA cleavage by the CAS9-crRNA complex is described by WO 2013/141680 A1 and WO 2013/142578 A1.
The CRISPR/Cas technology has been used to engineer gene knockouts in various mammalian cell types including diploid human cell lines (e.g. 293T cells) [15].
A near-haploid human cell line KBM-7 was reportedly used to inactivate single human genes using a retroviral gene trapping approach, thereby producing a collection of mutant KBM-7 cell lines carrying single gene trap insertions. Difficulties in producing a human library containing a knockout clone for each human gene have been described [16]. Such collections would be significantly distinct from the KBM-7 gene trap collections, in which the impact of the gene trap on gene expression is often incomplete and heavily dependent on the genomic locus of interest. TALENs (transcription activator-like effector nucleases) and CRISPR/Cas9 were used for genome engineering in a variety of cell types, including human cells [17].
Human knockout cells are invaluable tools that allow for the systematic investigation of human gene function in vitro. A collection of all human gene knockouts may be used, e.g. for reverse genetic studies or for the discovery of novel drug targets.
A prior art collection of human mutant cells was produced using gene-trap mutagenesis in near-haploid human cells. Every cell line carries a gene-trap insertion at a particular genomic locus, leading to the inactivation of that particular gene. In that regard, haploid gene trap mutants resemble conventional gene knockouts. Yet, gene traps cannot be targeted to a particular locus of interest and thus, the gene trap integration site is determined by the integration pattern of the retroviral vector used for the delivery of the gene trap. [12].
It is the object of the present invention to provide for an efficient method of producing somatic human cell lines with predefined mutations.